1. The Field of the Invention
This invention relates generally to stabilizing the position, temperature, and optical scattering characteristics of a biological sample undergoing optical evaluation.
2. Background and Relevant Art
Many forms of optical evaluation are being developed as noninvasive means of characterizing the chemistry and morphology of biological samples. In a number of applications it is desired to ascertain important properties of the tissue by spectroscopic analysis. For example, spectroscopy can be used to ascertain the concentration of an analyte of interest within the sample. In such cases, there may be variations in the chemical composition of the sample as a function of position (site to site variations). If the spectra of the chemicals which vary from site to site have some overlap with the spectrum of the analyte of interest, it is often difficult to obtain a calibration for the analyte concentration from multiple sites.
In addition, many important properties of the sample will change as a function of temperature. Examples include diffusion rates, vascular dilation, metabolic rates, and perspiration. A change in any of these properties can have significant effects on scattering and propagation of light through the material. In cases where lasers which deliver substantial power levels must be used to illuminate the sample, in order to obtain an adequate signal, the temperature increase caused by the laser radiation can interfere with the measurement. At sufficiently high powers the elevation of temperature caused by the laser can also damage or destroy proteins by denaturation, and cause a local burn to the skin of the subject.
In order to prevent excessive temperature excursions, it is useful to place the sample in contact with an optical window, the thermal conductivity of which is substantially higher than that of the sample. In such cases however, evaporation of volatile compounds such as water is impeded by the presence of the window. An illustrative example would be that of human skin where the deeper layers contain more water than the surface layers and when the evaporation from the surface is interrupted, moisture will diffuse into the outer layers and to the interface with the window. The progressive change of the moisture in these surface layers, and at the window interface, alters the scattering and reflection characteristics under optical illumination. The changing scattering and reflection characteristics may make it impossible to observe other changes that may be occurring on the same time scale. For example, if the concentration of some analyte in the skin is changing, the signal from spectroscopy for that analyte would be expected also to change, however, the changing scattering properties will also affect the size of the signal which is observed, and it can be exceptionally difficult to distinguish the two effects.
It is also observed that pressure on the sample can affect its optical properties, and fluids present in the sample can be physically moved by pressure. If the pressure is not fixed in time, the effects of changes in pressure can also be difficult to distinguish from changes in signal size associated with fluctuations in what is desired to be measured.
Finally, it is also the case that fluorescence from fluorophores present in the sample (autofluorescence), can interfere with certain forms of spectroscopic evaluation. For example, melanin is a strong fluorophore in human skin at excitation wavelengths in the visible range extending to the near IR range. If it is desired to obtain the Raman spectrum of an analyte of interest in human skin, the fluorescence induced by the laser, which is used to irradiate the sample, is usually the principle source of random noise in the measurement. In other cases, the fluorescence of one analyte is of interest, but the fluorescence of a second chemical which is also present in the sample is not of interest and is a source of unwanted interference. If one does not change the position of the sample during the course of the experiment, the analysis is generally simpler. Typically, the fluorescence will decay monotonically over time. This phenomenon is called photo-bleaching. If this process is interrupted by moving to a new spot on the sample, then the decay will start over again, thus complicating the analysis.